A basic enzyme-based biosensor electrode typically comprises a base conducting electrode system (carbon, gold, platinum, etc.) on an insulating substrate surmounted by a reagent layer containing at least a redox enzyme/cofactor (GDH/NAD, HBDH/NAD, GOx/FAD, GDH/PQQ, GDH/FAD) which acts on an analyte (glucose, 3-hydroxybutyrate, etc.) and a redox mediator which provides electrical communication between the enzyme/cofactor and the electrode. The action of enzyme/cofactor on the analyte results in the conversion of oxidized mediator to its reduced form which, in turn, is oxidized at an electrode. This generates an electrical signal which is proportional to the analyte concentration.
Commercial biosensor electrodes are manufactured on a large-scale using multi-stage processes of which reagent layer deposition is just one step. The reagent composition is required to be stable during manufacturing. Furthermore, the final packaged biosensor electrodes containing the dry reagent layer will be subjected to quality testing, shipping, distribution and storage such that it may be several months before the customer uses the product. Ideally, the shelf life of the biosensor electrode is in excess of 18 months from the date of manufacture. The stability of the biosensor electrode must be maintained during this period.
A common issue encountered by biosensor manufacturers is that the redox mediator, if in the oxidized form, tends to convert to the reduced form in the dry reagent layer over time (see, e.g., WO 2007/058999, EP 1398386). The problem is exacerbated in biamperometric systems where high concentrations of oxidized mediator are required for reduction by both the enzyme/cofactor and the reference electrode during the assay.
Furthermore, modern biosensor electrodes have fast assay times (5 seconds or less). This creates additional sensitivity of the reduced mediator in the reagent layer since it is immediately oxidized upon application of the electrode operating potential at the start of the assay. The result is an elevated background response, which varies over time such that the determination of low analyte concentrations in test samples can be inaccurate. Accordingly, there is a need in the art for methods and compositions that act to stabilize redox mediators in the presence of enzymes, when contained in dry reagent layers of biosensor electrodes. Such mediators may include, but are not limited to 1,2-quinone mediators, especially those containing 1,10-phenanthroline quinone (PQ), and more especially transition metal complexes of PQ.
1,2-Quinone compounds are known to be reactive toward nucleophiles via a variety of mechanisms. For example, U.S. Pat. No. 6,736,957 reports that many 1,2-quinones can react irreversibly with protein thiol groups leading to enzyme inactivation. This is avoided by PQ-type 1,2-quinones resulting in improved biosensor electrode stability (see, e.g., Forrow et al. (2005) Biosensors & Bioelectronics 20:1617-1625 for further discussion).
PQ-type mediators and other 1,2-quinones are also known to be susceptible to reduction by amines (see, e.g., Itoh et al. (1983) J. Am. Chem. Soc. 105:4431-4441). PQ-type mediator reaction with and consequent reduction by amines present in the biosensor reagent layer represents a major route toward conversion to the reduced mediator form.
This is due to the fact that amine functional groups are present in enzymes (e.g., lysine side-chains, terminal amino group), some stabilizers (e.g., proteins such as BSA, hydrolysed gelatin) and many buffer salts (e.g., TRIS, BES). Mediator reaction with enzyme amine groups may also lead to enzyme denaturation, cross-linking or inactivation (if the groups are involved in substrate or cofactor binding), i.e., another mode of biosensor electrode destabilization. FIG. 3 illustrates the mechanism of reaction of a PQ-type quinone with a primary amine (e.g., lysine side-chain) leading to the formation of a reduced aminoalcohol species, also pictured in the scheme below:
